Combination of a pd-1 antagonist and a raf inhibitor for treating cancer

ABSTRACT

Disclosed herein is a pharmaceutical combination for use in the prevention, delay of progression or treatment of cancer, wherein the pharmaceutical combination exhibits a synergistic efficacy. The pharmaceutical combination comprises a humanized antagonist monoclonal antibody against PD- and a RAF inhibitor. Also disclosed herein is a combination for use in the prevention, delay of progression or treatment of cancer in a subject, comprising administering to the subject a therapeutically effective amount of a humanized antagonist monoclonal antibody against PD-1 and a therapeutically effective amount of a RAF inhibitor.

This application claims the benefit of priority to International Application No. PCT/CN2016/088591 which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

Disclosed herein is a pharmaceutical combination for use in the prevention, delay of progression or treatment of cancer, wherein the pharmaceutical combination exhibits a synergistic efficacy. The pharmaceutical combination comprises a humanized antagonist monoclonal antibody against PD-1 and a RAF inhibitor. Also disclosed herein is a method for use in the prevention, delay of progression or treatment of cancer in a subject, comprising administering to the subject a therapeutically effective amount of a humanized antagonist monoclonal antibody against PD-1 and a therapeutically effective amount of a RAF inhibitor.

BACKGROUND OF THE INVENTION

Mitogen-activated protein kinase (MAPK) signaling pathway which consists of RAS-RAF-MEK-ERK kinase cascade is one of the critical signal transduction pathways in regulating diverse cellular activities, such as cell survival, growth, differentiation and proliferation. Genetic aberrations that lead to constitutive activation of the MAPK pathway are commonly observed in human cancers. Oncogenic B-Raf mutations, with V600E mutation accounting for at least 90%, have been detected in a variety of human malignancies. Inhibitors that selectively target mutated B-Raf such as vemurafenib and dabrafenib have achieved high response rate and been approved by FDA in treating melanoma patients harboring B-Raf^(V600E). Prior studies suggested that B-Raf-targeting therapies could increase antigen expression, decrease immune suppressive factors in tumor microenvironment, and improve homing of T effector cells to the tumors. Thus, selective B-Raf inhibitors were suggested to possess immunosensitization properties and could be used in combination with immunotherapies to achieve more durable disease control/response in treating cancer.

Programmed Death 1 protein (PD-1, Pdcd-1, or CD279) is a 55 KD receptor protein related to CD28/CTLA4 co-stimulatory/inhibitory receptor family (Blank et al., 2005 Cancer Immunol Immunother 54:307-314). The full length PD-1 contains 288 amino acid residues (NCBI accession number: NP_005009). Its extracellular domain consists of amino acid residues 1-167, and the cytoplasmic C-terminal tail comprises residues 191-288, which has two hypothetical immune-regulatory motifs, an immunoreceptor tyrosine-based inhibitory motif (ITIM; Vivier et al., 1997 Immunol Today 18:286-291) and an immunoreceptor tyrosine switch motif (ITSM; Chemnitz et al, 2004 J Immunol 173:945-954).

Two sequence-related ligands, PD-L1 (B7-H1) and PD-L2 (B7-DC), have been identified to specifically interact with PD-1, inducing intracellular signal transduction that inhibits CD3 and CD28 mediated T-cell activation (Riley, 2009 Immunol Rev 229: 114-125), which, in turn, attenuates T-cell activities, for example, reduction of cell proliferation, IL-2 and IFN-γ secretion, as well as other growth factor and cytokine secretion.

Expression of PD-1 was frequently found in immune cells such as T-cells, B-cells, monocytes and natural killer (NK) cells. It was rarely expressed in other human tissues, such as muscle, epithelium, neuronal tissues, etc. Furthermore, high level of PD-1 expression is often associated with activation of immune cells. For example, when human T-cell line, Jurkat, was activated by phytohaemagglutinin (PHA) or phorbol ester (12-0-tetradecanoylphorbol-13-acetate, or TP A), the expression of PD-1 was up-regulated visible in Western Blot (Vibharka et al., 1997 Exp Cell Res 232:25-28). The same phenomenon was observed in stimulated murine T- and B -lymphocytes and in primary human CD4⁺T-cells upon stimulation by anti-CD3 antibody (Agata et al., 1996 Int Immunol 8: 765-772; Bennett et al., 2003 J Immunol 170: 711-118). The increase of PD-1 expression following stimulation of T effector cells redirects the activated T-effector cells towards exhaustion and reduced immune activities. Therefore, PD-1 mediated inhibitory signal plays an important role in immune tolerance (Bour-Jordan et al., 2011 Immunol Rev 241:180-205).

Increase of PD-1 expression in tumor-infiltrating lymphocytes (TILs) and PD-1 ligand expression in tumor cells were reported in varieties of cancers involved in different types of tissues and organs such as lung (Konishi et al., 2004 Clin Cancer Res 10:5094-5100), liver (Shi et al, 2008 Int J Cancer 128:887-896; Gao et al, 2009 Clin Cancer Res 15:971- 979), stomach (Wu et al, 2006 Acta Histochem 108: 19-24), kidney (Thompson et al, 2004 Proc Natl Acad Sci 101:17174-17179; Thompson et al, 2007 Clin Cancer Res 13:1757-1761), breast (Ghebeh et al., 2006 Neoplasia 8: 190-198), ovary (Hamanishi et al. 2007 Proc Natl Acad Sci 104:3360-3365), pancreas (Nomi et al, 2007 Clin Cancer Res 13:2151-2157), melanocytes (Hino et al., 2010 Cancer 116: 1757-1766) and esophagus (Ohigashi et al., 2005 Clin Cancer Res 11:2947-2953). More frequently, the increased expression of PD-1 and PD-L1 in those cancers is associated with poor prognosis of patient survival outcome. Transgenic mice with PD-1 gene knockout inhibiting xenograft cancer cell growth further elucidated the significance of PD-1 signaling in the modulation of immune system for cancer eradication or tolerance (Zhang et al., 2009 Blood 114: 1545-1552).

WO 2013/097224 A1 disclosed a second generation B-RAF inhibitor, which has demonstrated potent inhibitory activity against RAF family of serine/threonine kinases.

PCT application PCT/CN2016/079251 discloses pharmaceutically-acceptable salt of the second generation B-RAF inhibitors in WO 2013/097224 A1, particularly, 5-(((1R,1aS,6bR)-1-(6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-1a,6b-dihydro-1H-cyclopropa[b]benzofuran-5-yl)oxy)-3,4-dihydro-1,8-naphthyrdin-2(1H)-one Sesqui-Maleate (hereinafter Compound 1) for the treatment of cancers with aberrations in the RAF-MEK-ERK MAPK pathway including B-Raf mutations, K-Ras/N-Ras mutations and NF1 mutations, which has potent and reversible inhibitory activities against RAF family kinases including A-Raf, B-Raf, C-Raf and B-Raf^(V600E).

WO 2015/035606 A1 disclosed a monoclonal antibody comprising a heavy chain variable region (Vh) and a light chain variable region (Vk) (comprising SEQ ID No 24 and SEQ ID No 26, respectively) and a IgG4 heavy chain effector or constant domain (comprising SEQ ID NOs: 88), hereinafter Mab 1, which specifically binds to PD-1, especially PD-1 residues including K45 and 193; or, 193, L95 and P97, and inhibits PD-1-medidated cellular signaling and activities in immune cells, antibodies binding to a set of amino acid residues required for its ligand binding.

The inventors of the present application have found that the combination of the above Anti-PD1 Antibodies (i.e., Mab 1) and antibody fragments thereof with the selective B-Raf inhibitor (i.e., Compound 1) surprisingly and unexpectedly augments T cell responses in a subject suffering from cancer associated with K-Ras mutations by enhancing IFN-γ production. In particular, the inventors of the present application have unexpectedly found that the combination of the particular Anti-PD1 Antibodies (i.e., Mab 1) and the particular selective B-Raf inhibitor (i.e., Compound 1) resulted in synergistic inhibition of tumor growth in cancers associated with K-Ras mutations as compared with the monotherapy of the Anti-PD1 Antibodies or the B-Raf inhibitor alone.

SUMMARY OF THE INVENTION

In a first aspect, disclosed herein is a pharmaceutical combination for use in the prevention, delay of progression or treatment of cancer, comprising a humanized antagonist monoclonal antibody against PD-1 and a RAF inhibitor. The pharmaceutical combination produces synergistic efficacy in inhibiting tumor growth in cancer.

In a second aspect, disclosed herein is a method for the prevention, delay of progression or treatment of cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a humanized antagonist monoclonal antibody against PD-1 and a therapeutically effective amount of a RAF inhibitor.

In a third aspect, disclosed herein is a humanized antagonist monoclonal antibody against PD-1 for use in the prevention, delay of progression or treatment of cancer in combination with a RAF inhibitor. In one embodiment of this aspect, disclosed herein is a RAF inhibitor for use in the prevention, delay of progression or treatment of cancer in combination with a humanized antagonist monoclonal antibody against PD-1.

In a fourth aspect, disclosed herein is a use of a pharmaceutical combination in the manufacture of a medicament for use in the prevention, delay of progression or treatment of cancer, said pharmaceutical combination comprising a humanized antagonist monoclonal antibody against PD-1 and a RAF inhibitor.

In a fifth aspect, disclosed herein is an article of manufacture, or “kit” comprising a first container, a second container and a package insert, wherein the first container comprises at least one dose of a medicament comprising a PD-1 antagonist, the second container comprises at least one dose of a medicament comprising a RAF inhibitor, and the package insert comprises instructions for treating cancer a subject using the medicaments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the level of IFN-y produced from activated PBMC in tumor spheriod/PBMC co-culture system after treatment with RAF inhibitor alone or combination of RAF inhibitor and anti-PD-1 mAb.

FIG. 2 shows the combination effect of RAF inhibitor and anti-PD-1 mAb on tumor growth in Calu-6 xenograft model in the presence of human PBMCs.

FIG. 3 shows the combination effect of RAF inhibitor and anti-PD-1 mAb on tumor growth in human primary colon cancer BCCO-028 xenograft model in the presence of human PBMC s.

FIG. 4 shows an X-ray diffraction pattern of Compound 1 in a crystalline form (crystallization from isopropanol/water).

FIG. 5 shows a ¹H-NMR spectrum of the crystalline form of Compound 1.

FIG. 6 shows a¹³C-NMR spectrum of the crystalline form of Compound 1.

DETAILED DESCRIPTION OF THE INVENTION Abbreviations

Throughout the detailed description and examples disclosed herein, the following abbreviations will be used:

CDR Complementarity determining region DPBS Dulbecco's Phosphate Buffered Saline DMEM Dulbecco minimum essential medium IgG immunoglobulin G i.p. Intraperitoneal or Intraperitoneally i.v. intravenous or intravenously IFN-γ Interferon-γ mAb Monoclonal antibodies MAPK Mitogen-activated protein kinase NK Natural killer PD-1 Programmed Death 1 protein, Pdcd-1, or CD279 PBMC Peripheral blood mononuclear cell PDX Patient-derived xenograft PHA Phytohaemagglutinin p.o. “by mouth” or “per os” QD Once daily QW Once weekly Q2W Once every two weeks Q3W Once every three weeks Q4W Once every four weeks TILs Tumor-infiltrating lymphocytes Vh Heavy chain variable region Vk Light chain variable region

Definitions

Unless specifically defined elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs.

As used herein, including the appended claims, the singular forms of words such as “a”, “an”, and “the”, include their corresponding plural references unless the context clearly dictates otherwise.

The term “about” used in the context of quantitative measurements means the indicated amount±20%, or alternatively the indicated amount±10% or ±5% or ±1%. For example, for Compound 1, a molar ratio (free base/maleic acid, n) of about 1 may vary between 0.8 and 1.2.

The term “alkyl” herein means a monoradical branched or linear saturated hydrocarbon chain comprising from 1 to 18, such as from 1 to 12, further such as from 1 to 6, carbon atoms. Alkyl groups include, but not limited to, methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, t-butyl, n-hexyl, n-decyl, tetradecyl, and the like.

The term “alkenyl” herein means a monoradical branched or linear unsaturated hydrocarbon group comprising at least one C═C double bond and from 2 to 18, such as from 2 to 8, further such as from 2 to 6 carbon atoms. Alkenyl groups include, but not limited to, ethenyl (or vinyl, i.e. —CH═CH₂), 1 -propylene (or allyl, i.e. —CH₂CH═CH₂), isopropylene (—C(CH₃)═CH₂), and the like.

The term “alkynyl” herein means a monoradical branched or linear unsaturated hydrocarbon group comprising at least one C≡C triple bond and from 2 to 18, such as from 2 to 8, further such as from 2 to 6 carbon atoms. Alkynyl groups include, but not limited to, ethynyl (—C≡CH), propargyl (or propynyl, i.e. —C≡CCH₃), and the like.

The term “cycloalkyl” herein means a cyclic alkyl group comprising from 3 to 20 carbon atoms, or from 3 to 10 carbon atoms, or from 3 to 8 carbon atoms or from 3 to 6 carbon atoms having a monocyclic ring or multiple condensed rings. A cycloalkyl group may be saturated and partially unsaturated. Examples of monocyclic saturated cycloalkyl groups include, but not limited to, C₃₋₈ cycloalkyl selected from the group consisting of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Examples of monocyclic partially unsaturated cycloalkyl groups include, but not limited to, cyclopent-l-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, 1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, and cyclohexadienyl.

The term “aryl” herein means a monovalent aromatic hydrocarbon radical comprising from 6 to 20 carbon atoms, such as from 6 to 10 carbon atoms derived by the removal of one hydrogen atom from a single carbon atom of a parent aromatic ring system. Aryl includes bicyclic radicals comprising an aromatic ring fused to a saturated, partially unsaturated ring, or aromatic carbocyclic or heterocyclic ring. Examples of aryl groups include, but not limited to, radicals derived from benzene (phenyl), substituted benzenes, naphthalene, anthracene, biphenyl, indenyl, indanyl, 1,2-dihydronapthalene, 1,2,3,4-tetrahydronapthyl, and the like.

The term “halogen” or “halo” herein means F, Cl, Br or I.

The term “heteroaryl” herein means a group selected from:

5- to 7-membered aromatic, monocyclic rings comprising at least one heteroatom, for example, from 1 to 4, or, in some embodiments, from 1 to 3, heteroatoms, selected from N, O, and S, with the remaining ring atoms being carbon;

8- to 12-membered bicyclic rings comprising at least one heteroatom, for example, from 1 to 4, or, in some embodiments, from 1 to 3, or, in other embodiments, 1 or 2, heteroatoms, selected from N, O, and S, with the remaining ring atoms being carbon and wherein at least one ring is aromatic and at least one heteroatom is present in the aromatic ring; and

11- to 14-membered tricyclic rings comprising at least one heteroatom, for example, from 1 to 4, or in some embodiments, from 1 to 3, or, in other embodiments, 1 or 2, heteroatoms, selected from N, O, and S, with the remaining ring atoms being carbon and wherein at least one ring is aromatic and at least one heteroatom is present in an aromatic ring.

For example, the heteroaryl group includes a 5- to 7-membered heterocyclic aromatic ring fused to a 5- to 7-membered cycloalkyl ring. For such fused, bicyclic heteroaryl ring systems wherein only one of the rings comprises at least one heteroatom, the point of attachment may be at the heteroaromatic ring or at the cycloalkyl ring.

When the total number of S and O atoms in the heteroaryl group exceeds 1, those heteroatoms are not adjacent to one another. In some embodiments, the total number of S and O atoms in the heteroaryl group is not more than 2. In some embodiments, the total number of S and O atoms in the aromatic heterocycle is not more than 1.

Examples of the heteroaryl group include, but not limited to, (as numbered from the linkage position assigned priority 1) pyridyl (such as 2-pyridyl, 3-pyridyl, or 4-pyridyl), cinnolinyl, pyrazinyl, 2,4-pyrimidinyl, 3,5-pyrimidinyl, 2,4-imidazolyl, imidazopyridinyl, isoxazolyl, oxazolyl, thiazolyl, isothiazolyl, thiadiazolyl, tetrazolyl, thienyl, triazinyl, benzothienyl, furyl, benzofuryl, benzoimidazolyl, indolyl, isoindolyl, indolinyl, phthalazinyl, pyrazinyl, pyridazinyl, pyrrolyl, triazolyl, quinolinyl, isoquinolinyl, pyrazolyl, pyrrolopyridinyl (such as 1H-pyrrolo[2,3-b]pyridin-5-yl), pyrazolopyridinyl (such as 1H-pyrazolo[3,4-b]pyridin-5-yl), benzoxazolyl (such as benzo[d]oxazol-6-yl), pteridinyl, purinyl, 1-oxa-2,3-diazolyl, 1-oxa-2,4-diazolyl, 1-oxa-2,5-diazolyl, 1-oxa-3,4-diazolyl, 1-thia-2,3-diazolyl, 1-thia-2,4-diazolyl, 1-thia-2,5-diazolyl, 1-thia-3,4-diazolyl, furazanyl, benzofurazanyl, benzothiophenyl, benzothiazolyl, benzoxazolyl, quinazolinyl, quinoxalinyl, naphthyridinyl, furopyridinyl, benzothiazolyl (such as benzo[d]thiazol-6-yl), indazolyl (such as 1H-indazol-5-yl) and 5,6,7,8-tetrahydroisoquinoline.

The term “heterocyclic” or “heterocycle” or “heterocyclyl” herein means a ring selected from 4- to 12-membered monocyclic, bicyclic and tricyclic, saturated and partially unsaturated rings comprising at least one carbon atoms in addition to at least one heteroatom, such as from 1-4 heteroatoms, further such as from 1-3, or further such as 1 or 2 heteroatoms, selected from oxygen, sulfur, and nitrogen. “Heterocycle” herein also means a 5- to 7-membered heterocyclic ring comprising at least one heteroatom selected from N, O, and S fused with 5-, 6-, and/or 7-membered cycloalkyl, carbocyclic aromatic or heteroaromatic ring, provided that the point of attachment is at the heterocyclic ring when the heterocyclic ring is fused with a carbocyclic aromatic or a heteroaromatic ring, and that the point of attachment can be at the cycloalkyl or heterocyclic ring when the heterocyclic ring is fused with cycloalkyl. “Heterocycle” herein also means an aliphatic spirocyclic ring comprising at least one heteroatom selected from N, O, and S, provided that the point of attachment is at the heterocyclic ring. The rings may be saturated or have at least one double bond (i.e., partially unsaturated). The heterocycle may be substituted with oxo. The point of the attachment may be carbon or heteroatom in the heterocyclic ring. A heterocycle is not a heteroaryl as defined herein.

Examples of the heterocycle include, but not limited to, (as numbered from the linkage position assigned priority 1) 1-pyrrolidinyl, 2-pyrrolidinyl, 2,4-imidazolidinyl, 2,3-pyrazolidinyl, 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 2,5-piperazinyl, pyranyl, 2-morpholinyl, 3-morpholinyl, oxiranyl, aziridinyl, thiiranyl, azetidinyl, oxetanyl, thietanyl, 1,2-dithietanyl, 1,3-dithietanyl, dihydropyridinyl, tetrahydropyridinyl, thiomorpholinyl, thioxanyl, piperazinyl, homopiperazinyl, homopiperidinyl, azepanyl, oxepanyl, thiepanyl, 1,4-oxathianyl, 1,4-dioxepanyl, 1,4-oxathiepanyl, 1,4-oxaazepanyl, 1,4-dithiepanyl, 1,4-thiazepanyl and 1,4-diazepane 1,4-dithianyl, 1,4-azathianyl, oxazepinyl, diazepinyl, thiazepinyl, dihydrothienyl, dihydropyranyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, 1-pyrrolinyl, 2-pyrrolinyl, 3-pyrrolinyl, indolinyl, 2H-pyranyl, 4H-pyranyl, 1,4-dioxanyl, 1,3-dioxolanyl, pyrazolinyl, pyrazolidinyl, dithianyl, dithiolanyl, pyrazolidinyl, imidazolinyl, pyrimidinonyl, 1,1-dioxo-thiomorpholinyl, 3-azabicyco[3,1,0]hexanyl, 3-azabicyclo[4,1,0]heptanyl and azabicyclo[2,2,2]hexanyl. A substituted heterocycle also includes a ring system substituted with one or more oxo moieties, such as piperidinyl N-oxide, morpholinyl-N-oxide, 1-oxo-1-thiomorpholinyl and 1,1-dioxo-1-thiomorpholinyl.

The term “fused ring” herein means a polycyclic ring system, e.g., a bicyclic or tricyclic ring system, in whcih two rings share only two ring atoms and one bond in common. Examples of fused rings may comprise a fused bicyclic cycloalkyl ring such as those having from 7 to 12 ring atoms arranged as a bicyclic ring selected from [4,4], [4,5], [5,5], [5,6] and [6,6] ring systems as mentioned above; a fused bicylclic aryl ring such as 7 to 12 membered bicyclic aryl ring systems as mentioned above, a fused tricyclic aryl ring such as 10 to 15 membered tricyclic aryl ring systems mentioned above; a fused bicyclic heteroaryl ring such as 8- to 12-membered bicyclic heteroaryl rings as mentioned above, a fused tricyclic heteroaryl ring such as 11- to 14-membered tricyclic heteroaryl rings as mentioned above; and a fused bicyclic or tricyclic heterocyclyl ring as mentioned above.

The terms “administration”, “administering”, “treating” and “treatment” herein, when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, mean contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition to the animal, human, subject, cell, tissue, organ, or biological fluid. Treatment of a cell encompasses contact of a reagent to the cell, as well as contact of a reagent to a fluid, where the fluid is in contact with the cell. The term “administration” and “treatment” also means in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell. The term “subject” herein includes any organism, preferably an animal, more preferably a mammal (e.g., rat, mouse, dog, cat, rabbit) and most preferably a human.

The term “pharmaceutically acceptable salt” herein includes, but not limited to salts with inorganic acids, selected, for example, from hydrochlorates, phosphates, diphosphates, hydrobromates, sulfates, sulfinates, and nitrates; as well as salts with organic acids, selected, for example, from malates, maleates, fumarates, tartrates, succinates, citrates, lactates, methanesulfonates, p- toluenesulfonates, 2-hydroxyethylsulfonates, benzoates, salicylates, stearates, alkanoates such as acetate, and salts with HOOC—(CH₂)_(n)—COOH, wherein n is selected from 0 to 4. Similarly, examples of pharmaceutically acceptable cations include, but not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium.

The term “antibody” herein is used in the broadest sense and specifically covers antibodies (including full length monoclonal antibodies) and antibody fragments so long as they recognize PD-1. An antibody molecule is usually monospecific, but may also be described as idiospecific, heterospecific, or polyspecific. Antibody molecules bind by means of specific binding sites to specific antigenic determinants or epitopes on antigens. “Antibody fragments” comprise a portion of a full length antibody, generally the antigen binding or variable region thereof. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The term “monoclonal antibody” or “mAb” or “Mab” herein means a population of substantially homogeneous antibodies, i.e., the antibody molecules comprised in the population are identical in amino acid sequence except for possible naturally occurring mutations that may be present in minor amounts. In contrast, conventional (polyclonal) antibody preparations typically include a multitude of different antibodies having different amino acid sequences in their variable domains, particularly their CDRs, which are often specific for different epitopes. The modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. Monoclonal antibodies (mAbs) may be obtained by methods known to those skilled in the art. See, for example Kohler et al (1975); U.S. Pat. No. 4,376,110; Ausubel et al (1987-1999); Harlow et al (1988); and Colligan et al (1993). The mAbs disclosed herein may be of any immunoglobulin class including IgG, IgM, IgD, IgE, IgA, and any subclass thereof. A hybridoma producing a mAb may be cultivated in vitro or in vivo. High titers of mAbs can be obtained in in vivo production where cells from the individual hybridomas are injected intraperitoneally into mice, such as pristine-primed Balb/c mice to produce ascites fluid containing high concentrations of the desired mAbs. MAbs of isotype IgM or IgG may be purified from such ascites fluids, or from culture supernatants, using column chromatography methods well known to those of skill in the art.

In general, the basic antibody structural unit comprises a tetramer. Each tetramer includes two identical pairs of polypeptide chains, each pair having one “light chain” (about 25 kDa) and one “heavy chain” (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of the heavy chain may define a constant region primarily responsible for effector function. Typically, human light chains are classified as kappa and lambda light chains. Furthermore, human heavy chains are typically classified as α, δ, ε, γ, or μ, and define the antibody's isotypes as IgA, IgD, IgE, IgG, and IgM, respectively. Within light and heavy chains, the variable and constant regions are joined by a “J” region of about 12 or more amino acids, with the heavy chain also including a “D” region of about 10 more amino acids.

The variable regions of each light/heavy chain (Vk/Vh) pair form the antibody binding site. Thus, in general, an intact antibody has two binding sites. Except in bifunctional or bispecific antibodies, the two binding sites are, in general, the same.

Typically, the variable domains of both the heavy and light chains comprise three hypervariable regions, also called “complementarity determining regions (CDRs)”, which are located within relatively conserved framework regions (FR). The CDRs are usually aligned by the framework regions, enabling binding to a specific epitope. In general, from N-terminal to C-terminal, both light and heavy chains variable domains comprise FR-1, CDR-1, FR-2, CDR-2, FR-3, CDR-3, and FR-4. The assignment of amino acids to each domain is, generally, in accordance with the definitions of Sequences of Proteins of Immunological Interest, Kabat, et al. National Institutes of Health, Bethesda, Md. ; 5<m>ed.; NIH Publ. No. 91-3242 (1991); Kabat (1978) Adv. Prot. Chem. 32: 1-75; Kabat, et al., (1977) J. Biol. Chem. 252:6609-6616; Chothia, et al, (1987) J Mol. Biol. 196:901-917 or Chothia, et al, (1989) Nature 342:878-883.

The term “hypervariable region” means the amino acid residues of an antibody that are responsible for antigen-binding. The hypervariable region comprises amino acid residues from a “complementarity determining region” or “CDR” (i.e., CDR-L1, CDR-L2 and CDR-L3 in the light chain variable domain and CDR-H1, CDR-H2 and CDR-H3 in the heavy chain variable domain). See, Kabat et al. (1991) Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (defining the CDR regions of an antibody by sequence); see also Chothia and Lesk (1987) J. Mol. Biol. 196: 901-917 (defining the CDR regions of an antibody by structure). The term “framework” or “FR” residues means those variable domain residues other than the hypervariable region residues defined herein as CDR residues.

Unless otherwise indicated, “antibody fragment” or “antigen binding fragment” means antigen binding fragments of antibodies, i.e. antibody fragments that retain the ability to bind specifically to the antigen bound by the full-length antibody, e.g. fragments that retain one or more CDR regions. Examples of antibody binding fragments include, but not limited to, Fab, Fab′, F(ab′)2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules, e.g., sc-Fv; nanobodies and multispecific antibodies formed from antibody fragments.

An antibody that “specifically binds to” a specified target protein is an antibody that exhibits preferential binding to that target as compared to other proteins, but this specificity does not require absolute binding specificity. An antibody is considered “specific” for its intended target if its binding is determinative of the presence of the target protein in a sample, e.g. without producing undesired results such as false positives. Antibodies, or binding fragments thereof, useful in the present invention will bind to the target protein with an affinity that is at least two fold greater, preferably at least ten times greater, more preferably at least 20-times greater, and most preferably at least 100-times greater than the affinity with non-target proteins. An antibody herein is said to bind specifically to a polypeptide comprising a given amino acid sequence, e.g. the amino acid sequence of a mature human PD-1 molecule, if it binds to polypeptides comprising that sequence but does not bind to proteins lacking that sequence.

The term “human antibody” herein means an antibody that comprises human immunoglobulin protein sequences only. A human antibody may contain murine carbohydrate chains if produced in a mouse, in a mouse cell, or in a hybridoma derived from a mouse cell. Similarly, “mouse antibody” or “rat antibody” mean an antibody that comprises only mouse or rat immunoglobulin sequences, respectively.

The term “humanized antibody” means forms of antibodies that contain sequences from non-human (e.g., murine) antibodies as well as human antibodies. Such antibodies contain minimal sequence derived from non-human immunoglobulin. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. The prefix “hum”, “hu” or “h” is added to antibody clone designations when necessary to distinguish humanized antibodies from parental rodent antibodies. The humanized forms of rodent antibodies will generally comprise the same CDR sequences of the parental rodent antibodies, although certain amino acid substitutions may be included to increase affinity, increase stability of the humanized antibody, or for other reasons.

The terms “cancer” or “tumor” herein mean or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Examples of cancer include but not limited to, adrenal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer (including small-cell lung cancer, or non-small cell lung cancer), lymphoma, melanoma, ovarian cancer, pancreatic cancer, skin cancer, or thyroid tumors and their complications. Particularly preferred cancers or tumors that may be treated by the combination disclosed herein include those characterized by B-Raf mutations, K-Ras/N-Ras mutations and/or NF1 mutations. The most preferred cancers or tumors that may be treated by the combination disclosed herein include non-small cell lung cancer, colorectal cancer, and endometrial cancer, each of which is associated with K-Ras mutations.

The term “CDRs” means complementarity determining region(s) in an immunoglobulin variable region, defined using the Kabat numbering system, unless otherwise indicated.

“PD-1 antagonist” means any chemical compound or biological molecule that blocks binding of PD-L1 expressed on a cancer cell to PD-1 expressed on an immune cell (T cell, B cell or NKT cell) and preferably also blocks binding of PD-L2 expressed on a cancer cell to the immune-cell expressed PD-1. Alternative names or synonyms for PD-1 and its ligands include: PDCD1, PD1, CD279 and SLEB2 for PD-1; PDCD1L1, PDL-1, B7H1, B7-4, CD274 and B7-H for PD-L1; and PDCD1L2, PDL2, B7-DC, Btdc and CD273 for PD-L2.

PD-1 Antagonist

As disclosed in each of the above five aspects, the PD-1 antagonist is an antibody or a fragment antigen binding thereof, which specifically binds to human PD-1.

As disclosed in each of the above five aspects, the PD-1 antagonist is an antibody which comprises a heavy chain variable region (Vh) and a light chain variable region (Vk) that contain complement determinant regions (CDRs) listed as follows:

a) mu317 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 11, 12, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 14, 15, 16, respectively); b) mu326 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 17, 18, 19, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 20, 21, 22, respectively); c) 317-4B6 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 31, 32, 33, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 34, 35, 36, respectively); d) 326-4A3 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 37, 38, 39, respectively); and CDR-L1, CDR--L2 and CDR-L3 (SEQ ID NOs: 40, 41, 42, respectively); e) 317-1H CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 11, 59, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 14, 15, 16, respectively); f) 317-4B2 CDR-HL CDR-H2 and CDR-H3 (SEQ ID NOs: 11, 60, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 61, 15, 16, respectively); g) 317-4B5 CDR-Hl, CDR-H2 and CDR-H3 (SEQ ID NOs: 11, 60, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 61, 15, 16, respectively); h) 317-4B6 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 11, 32, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 61, 15, 16, respectively); i) 326-1 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 17, 62, 19, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 20, 21, 22, respectively); j) 326-3B1 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 17, 62, 19, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 20, 21, 22, respectively); or k) 326-3G1 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 17, 62, 19, respectively); and CDR-L1, CDR-I2 and CDR-L3 (SEQ ID NOs: 20, 21, 22, respectively).

As disclosed in each of the above five aspects, the PD-1 antagonist is an antibody which comprises a heavy chain variable region (Vh) and a light chain variable region (Vk) that contain any combinations of CDRs listed as follows:

(a) CDR-H1 (SEQ ID NO 31), CDR-H2 (SEQ ID NO 12, 32, 59 or 60) and CDR-H3 (SEQ ID NO 33), CDR-L1 (SEQ ID NO 14, 34 or 61), CDR-L2 (SEQ ID NO 35) and CDR-L3 (SEQ ID NO 36); or (b) CDR-H1 (SEQ ID NO 37), CDR-H2 (SEQ ID NO 18, 38 or 62) and CDR-H3 (SEQ ID NO 39), CDR-L1 (SEQ ID NO 40), CDR-L2 (SEQ ID NO 41) and CDR-L3 (SEQ ID NO 42).

As disclosed in each of the above five aspects, the PD-1 antagonist is an antibody which comprises a heavy chain variable region (Vh) and a light chain variable region (Vk) comprising:

a) mu317 (SEQ ID NOs: 4 and 6, respectively); b) mu326 (SEQ ID NOs: 8 and 10, respectively); c) 317-4B6 (SEQ ID NOs: 24 and 26, respectively); d) 326-4A3 (SEQ ID NOs: 28 and 30, respectively); e) 317-4B2 (SEQ ID NOs: 43 and 44, respectively); f) 317-4B5 (SEQ ID NOs: 45 and 46, respectively); g) 317-1 (SEQ ID NOs: 48 and 50, respectively); h) 326-3B1 (SEQ ID NOs: 51 and 52, respectively); i) 326-3GI (SEQ ID NOs: 53 and 54, respectively); j) 326-1 (SEQ ID NOs: 56 and 58, respectively); k) 317-3A1 (SEQ ID NOs: 64 and 26, respectively); l) 317-3C1 (SEQ ID NOs: 65 and 26, respectively); m) 317-3E1 (SEQ ID NOs: 66 and 26, respectively); n) 317-3F1 (SEQ ID NOs: 67 and 26, respectively); o) 317-3G1 (SEQ ID NOs: 68 and 26, respectively); p) 317-3H1 (SEQ ID NOs: 69 and 26, respectively); q) 317-311 (SEQ ID NOs: 70 and 26, respectively); r) 317-4B1 (SEQ ID NOs: 71 and 26, respectively); s) 317-4B3 (SEQ ID NOs: 72 and 26, respectively); t) 317-4B4 (SEQ ID NOs: 73 and 26, respectively); u) 317-4A2 (SEQ ID NOs: 74 and 26, respectively); v) 326-3A1 (SEQ ID NOs: 75 and 30, respectively); w) 326-3C1 (SEQ ID NOs: 76 and 30, respectively); x) 326-3D1 (SEQ ID NOs: 77 and 30, respectively); y) 326-3E1 (SEQ ID NOs: 78 and 30, respectively); z) 326-3F1 (SEQ ID NOs: 79 and 30, respectively); aa) 326-3B N55D (SEQ ID NOs: 80 and 30, respectively); ab) 326-4A1 (SEQ ID NOs: 28 and 81, respectively); or ac) 326-4A2 (SEQ ID NOs: 28 and 82, respectively).

As disclosed in each of the above five aspects, the PD-1 antagonist is an antibody which comprises a IgG4 heavy chain effector or constant domain comprising any of SEQ ID NOs: 83-88.

As disclosed in each of the above five aspects, the PD-1 antagonist is an antibody which contains a F(ab) or F(ab)₂ comprising a domain said above, including a heavy chain variable region (Vh), a light chain variable region (Vk) and a IgG4 heavy chain effector or constant domain.

As disclosed in each of the above five aspects, the PD-1 antagonist is an antibody which comprise a heavy chain variable region (Vh) and a light chain variable region (Vk), and a IgG4 heavy chain effector or constant domain comprising SEQ ID NOs: 87 or 88, wherein the heavy chain variable region (Vh) and the light chain variable region (Vk) comprise:

a) mu317 (SEQ ID NOs: 4 and 6, respectively); b) mu326 (SEQ ID NOs: 8 and 10, respectively); c) 317-4B6 (SEQ ID NOs: 24 and 26, respectively); d) 326-4A3 (SEQ ID NOs: 28 and 30, respectively); e) 317-4B2 (SEQ ID NOs: 43 and 44, respectively); f) 317-4B5 (SEQ ID NOs: 45 and 46, respectively); g) 317-1 (SEQ ID NOs: 48 and 50, respectively); h) 326-3B1 (SEQ ID NOs: 51 and 52, respectively); i) 326-3GI (SEQ ID NOs: 53 and 54, respectively); j) 326-1 (SEQ ID NOs: 56 and 58, respectively); k) 317-3A1 (SEQ ID NOs: 64 and 26, respectively); l) 317-3C1 (SEQ ID NOs: 65 and 26, respectively); m) 317-3E1 (SEQ ID NOs: 66 and 26, respectively); n) 317-3F1 (SEQ ID NOs: 67 and 26, respectively); o) 317-3G1 (SEQ ID NOs: 68 and 26, respectively); p) 317-3H1 (SEQ ID NOs: 69 and 26, respectively); q) 317-311 (SEQ ID NOs: 70 and 26, respectively); r) 317-4B1 (SEQ ID NOs: 71 and 26, respectively); s) 317-4B3 (SEQ ID NOs: 72 and 26, respectively); t) 317-4B4 (SEQ ID NOs: 73 and 26, respectively); u) 317-4A2 (SEQ ID NOs: 74 and 26, respectively); v) 326-3A1 (SEQ ID NOs: 75 and 30, respectively); w) 326-3C1 (SEQ ID NOs: 76 and 30, respectively); x) 326-3D1 (SEQ ID NOs: 77 and 30, respectively); y) 326-3E1 (SEQ ID NOs: 78 and 30, respectively); z) 326-3F1 (SEQ ID NOs: 79 and 30, respectively); aa) 326-3B N55D (SEQ ID NOs: 80 and 30, respectively); ab) 326-4A1 (SEQ ID NOs: 28 and 81, respectively); or ac) 326-4A2 (SEQ ID NOs: 28 and 82, respectively).

As disclosed in each of the above five aspects, the PD-1 antagonist is an antibody which comprises a heavy chain variable region (Vh) and a light chain variable region (Vk), and an IgG4 heavy chain effector or constant domain comprising SEQ ID NO: 88, wherein the heavy chain variable region (Vh) and the light chain variable region (Vk) comprises SEQ ID NO: 24 and SEQ ID NO: 26, respectively.

The Anti-PD1 Antibodies and antibody fragments thereof disclosed herein may be prepared in accordance with the disclosure of WO 2015/035606 A1, the entire disclosure of which is expressly incorporated herein by reference.

RAF inhibitors

“RAF inhibitor” means a compound of Formula (I), or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

As disclosed in each of the above five aspects, the RAF inhibitor is a compound of Formula (I),

or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein:

X is selected from CH₂ and O;

R⁸, R⁹, R¹⁰ and R¹¹, which may be the same or different, are each selected from hydrogen, halogen, alkyl, alkenyl, cycloalkyl, aryl, heterocyclyl, heteroaryl, alkynyl, —NR¹³R¹⁴, —OR¹³, —COR¹³, —CO₂R¹³, —CONR¹³R¹⁴, —C(═NR¹³)NR¹⁴R¹⁵, —NR¹³COR¹⁴, —NR¹³CONR¹⁴R¹⁵, —NR¹³CO₂R¹⁴, —SO₂R¹³, —SO₂aryl, —NR¹³SO₂NR¹⁴R¹⁵, and —NR¹³SO₂R¹⁴, wherein the alkyl, alkenyl, alkynyl, cycloalkyl, heteroaryl, aryl, and heterocyclyl are each optionally substituted with at least one substituent R^(16,) or (R⁸ and R⁹), and/or (R⁹ and R¹⁰), and/or (R¹⁰ and R¹¹) together with the ring to which they are attached, form a fused ring selected from heterocyclyl, and heteroaryl rings optionally substituted with at least one substituent R¹⁶;

R¹³, R¹⁴ and R¹⁵, which may be the same or different, are each selected from H, haloalkyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl; or (R¹³ and R¹⁴), and/or (R¹⁴ and R¹⁵) together with the atom(s) to which they are attached, each form a ring selected from heterocyclyl, and heteroaryl rings optionally substituted with at least one substituent R¹⁶;

R¹⁶ is selected from halogen, haloalkyl, alkyl, alkenyl, cycloalkyl, aryl, heteroaryl, heterocyclyl, alkynyl, oxo, —CN, —OR′, —NR′R″, —COR′, —CO₂R′, —CONR′R″, —C(═NR′)NR″R′″, —NR′COR″, —NR′CONR′R″, —NR′CO₂R″, —SO₂R′, —SO₂aryl, —NR′S O₂NR″R′″, —NR′O₂R″, and —NR′SO₂aryl, wherein R′, R″, and R′″ are independently selected from H, haloalkyl, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl, or (R′ and R″), and/or (R″ and R′″) together with the atoms to which they are attached, form a ring selected from heterocyclyl, and heteroaryl rings.

In some embodiments, the compound of Formula (I) is optically pure.

In some embodiments, X in Formula (I) is O.

In some embodiments, X in Formula (I) is CH₂.

In some embodiments, R⁸, R⁹, R¹⁰, and R¹¹ in Formula (I), which may be the same or different, are each independently selected from alkyl (e.g., methyl, tert-butyl), hydrogen, haloalkyl (e.g., —CF₃), halogen, hydroxy, —CN, -Oalkyl (e.g., methoxy), -Ohaloalkyl (e.g., OCF₃), and aryl (e.g., phenyl).

As disclosed in each of the above five aspects, the RAF inhibitor is a compound of Formula (I),

or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein:

X is selected from CH₂ and O;

R⁸, R⁹, R¹⁰ and R¹¹, which may be the same or different, are each selected from hydrogen, halogen, alkyl, —CN, cycloalkyl, aryl, heterocyclyl, —OR¹³, —CONR¹³R¹⁴, wherein the alkyl, and aryl are each optionally substituted with at least one substituent R¹⁶, or (R⁸ and R⁹), and/or (R⁹ and R¹⁰), and/or (R¹⁰ and R¹¹) together with the ring to which they are attached, form a fused ring selected from cycloalkyl;

R¹³, and R¹⁴, which may be the same or different, are each selected from H, alkyl, and haloaklyl;

R¹⁶ is selected from halogen, haloalkyl, and alkyl.

As disclosed in each of the above five aspects, the RAF inhibitor is a compound of Formula (I),

or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein:

X is selected from CH₂ and O;

R⁸ is selected from H, F, Cl, and Br;

R⁹ is selected from H, F, Cl, Br, and C₁₋₆alkyl;

R¹⁰ is selected from H, F, Cl, Br, OH, —CN, C₁₋₆alkyl, CF₃, phenyl, OC₁₋₆alkyl, OC₁₋₆haloalkyl, and —CONR¹³R¹⁴, wherein R¹³ and R¹⁴ may be the same or different, are each selected from H, and C₁₋₆alkyl;

R¹¹ is selected from H, F, Cl, Br, and CF₃;

or (R⁹ and R¹⁰) together with the ring to which they are attached, form a fused ring selected from C₅₋₆cycloalky.

As disclosed in each of the above five aspects, the RAF inhibitor is a compound of selected from the following compounds:

or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof.

As disclosed in each of the above five aspects, the RAF inhibitor is a compound of selected from the following compounds:

or a stereoisomer thereof, or a pharmaceutically acceptable salt thereof

As disclosed in each of the above five aspects, the RAF inhibitor is a compound of selected from the following compounds:

or a pharmaceutically acceptable salt thereof.

As disclosed in each of the above five aspects, the RAF inhibitor is a compound of Formula (II),

or a pharmaceutically acceptable salt thereof.

As disclosed in each of the above five aspects, the RAF inhibitor is a maleate salt of the compound of Formula (II).

As disclosed in each of the above five aspects, the RAF inhibitor is a compound of Formula (III),

wherein n is a number from about 0.5 to about 1.5.

As disclosed in each of the above five aspects, the RAF inhibitor is a compound of Formula (IIIa)—i.e., Compound 1,

The RAF inhibitor disclosed herein, such as the compound of Formula (I), may be synthesized by synthetic routes disclosed in WO 2013/097224 A1, the entire disclosure of which is expressly incorporated herein by reference. The RAF inhibitor, i.e., Compound 1, disclosed herein, may be prepared in accordance with the procedures in PCT/CN2016/079251, the entire disclosure of which is expressly incorporated herein by reference.

Combination Therapy

The combination therapy may be administered as a simultaneous, or separate or sequential regimen. When administered sequentially, the combination may be administered in two or more administrations. The combined administration includes coadministration, using separate formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.

In one embodiment of each of the above five aspects, the RAF inhibitor or the pharmaceutically acceptable salt thereof can be administered for a time period of about 1 to about 10 days after administration of the PD-1 antagonist. In another embodiment of each of the above five aspects, the RAF inhibitor or the pharmaceutically acceptable salt thereof can be administered for a time period of about 1 to 10 days before administration of the combination begins. In another embodiment of each of the above five aspects, administration of the RAF inhibitor or the pharmaceutically acceptable salt thereof and administration of the PD-1 antagonist begin on the same day.

Suitable dosages for any of the above coadministered agents are those presently used and may be lowered due to the combined action (synergy) of the RAF inhibitor and the PD-1 antagonist, such as to increase the therapeutic index or mitigate toxicity or other side-effects or consequences.

In a particular embodiment of anti-cancer therapy, the RAF inhibitor and the PD-1 antagonist may be further combined with surgical therapy and radiotherapy.

In an embodiment of each of the above five aspects, the amounts of the RAF inhibitor and the PD-1 antagonist disclosed herein and the relative timings of administration be determined by the individual needs of the patient to be treated, administration route, severity of disease or illness, dosing schedule, as well as evaluation and judgment of the designated doctor.

For example, the administered dosage of the RAF inhibitor is 1-100 mg/day (in terms of the parent compound), and the administration frequency is one to three times a day; preferably, the administered dosage of the RAF inhibitor is 5-80 mg/day (in terms of the parent compound), and the administration frequency is one to three times a day; more preferably, the administered dosage of the RAF inhibitor is 10-40 mg/day (in terms of the parent compound), and the administration frequency is one time a day. However, based on the active compound, the preferred range of the effective dosage of the RAF inhibitor disclosed herein may be approximately 0.01-10 mg daily per kilogram of body weight; or more preferably 0.1-1 mg per day per kilo gram of body weight in single or separate doses. In some cases, it is more suitable to apply the lower end of the above described dosage ranges, while in other cases the higher dosages may be used without causing harmful side effects. In some preferred embodiment of each of the above five aspects, the RAF inhibitor is administrated at a dose of 5-80 mg once daily.

The PD-1 antagonist is administered at a dose of 0.5-30 mg/kg, such as 0.5-20 mg/kg, further such as 0.5-10 mg/kg once weekly, or every two weeks, or every three weeks , or every four weeks.

The RAF inhibitor and the PD-1 antagonist disclosed herein may be administered in various known manners, such as orally, topically, rectally, parenterally, by inhalation spray, or via an implanted reservoir, although the most suitable route in any given case will depend on the particular host, and nature and severity of the conditions for which the active ingredient is being administered. The term “parenteral” as used herein includes subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

In one embodiment of each of the above five aspects, the RAF inhibitor and the PD-1 antagonist disclosed herein may be administered in different route. In a preferred embodiment, the RAF inhibitor is administered orally, and the PD-1 antagonist is administered parenterally such as subcutaneously, intracutaneously, intravenously or intraperitoneally.

In an embodiment of each of the above five aspects, the PD-1 antagonist is an antibody which comprises a heavy chain variable region (Vh) and a light chain variable region (Vk), and a IgG4 heavy chain effector or constant domain comprising SEQ ID NO: 88, wherein the heavy chain variable region (Vh) and the light chain variable region (Vk) comprise SEQ ID NO: 24 and SEQ ID NO: 26, respectively; and the RAF inhibitor is the compound of Formula (IIIa) disclosed herein.

In an embodiment of each of the above five aspects, the PD-1 antagonist Mab-1 is administrated to a subject at a dose of 0.5-10 mg/kg i.v. or i.p. QW or Q2W or Q3W, and the RAF inhibitor Compound 1 is administrated to a subject at a dose of 5-80 mg QD. In some preferred embodiments, the PD-1 antagonist Mab-1 is administrated to a subject at a dose of 0.5-10 mg/kg i.v. or i.p. QW or Q2W or Q3W, and the RAF inhibitor Compound 1 is administrated to a subject at a dose of 10-30 mg QD. In an even more preferred embodiment, the PD-1 antagonist Mab-1 is administrated parenterally such as subcutaneously, intracutaneously, intravenously or intraperitoneally.

Methods of Treatment

The pharmaceutical combination produces synergistic efficacy in inhibiting tumor growth in cancer, such as adrenal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer (including small-cell lung cancer, or non-small cell lung cancer), lymphoma, melanoma, ovarian cancer, pancreatic cancer, skin cancer, or thyroid tumors and their complications. Particularly, the combination is effective in the above cancer associated with B-Raf mutations, K-Ras/N-Ras mutations and/or NF1 mutations. The most preferred cancers or tumors that may be treated by the combination disclosed herein include non-small cell lung cancer, colorectal cancer, and endometrial cancer, each of which is associated with K-Ras mutations. The combination is useful in a method for the prevention, delay of progression or treatment of cancer.

EXAMPLES Example A Preparation of Compound 1 (i.e., the Sesqui-Maleate Salt) Step 1: Synthesis of Intermediate 1

To a stirred solution of EtONa (154 kg) in DMF (989 kg) was added EtSH(68.6 kg) at an inner temperature ≤35° C. under nitrogen protection. The mixture was stirred for 60˜90min at the inner temperature ≤35° C. 5-Methoxybenzofuran (58.75 kg) in DMF (55.0 kg) was added. The mixture was heated to 110-130° C., stirred for 45 hours, and then concentrated under vacuum below 90° C. After the mixture was cooled to 10˜20° C., 2N HCl (1326 kg) was added dropwise, followed by addition of EtOAc (531 kg) and H₂O₂ (129 kg) at the inner temperature ≤35° C. The mixture was stirred for 30˜60min. After separation of the organic layer, the aqueous phase was extracted with EtOAc. The combined organic phase was washed with saturated brine twice, and then the solvent was evaporated to dryness. MeOH and a solution of NaOH (44.5 kg) in water (185 kg) were added dropwise into the residue below 40° C. The mixture was stirred for 5-7 hours at 30˜40° C. Active carbon (74 kg) wet up with water (77 kg) was added. The mixture was stirred for 4-6 hours at 30˜40° C. and filtered; and the filter cake was washed with MeOH and water. DCM was charged into the filtrate and pH was adjusted to 1 with 35% aq. HCl below 40° C. The aqueous phase was extracted with DCM, and the organic phase was washed with 25% NaCl and concentrated below 40° C. The residue was used in the next step directly. ¹H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H), 7.86 (d, J=2.0 Hz, 1H), 7.36 (d, J=8.8 Hz, 1H), 6.94 (d, J=2.4 Hz, 1H), 6.79 (dd, J=2.0, 0.9 Hz, 1H), 6.74 (dd, J=8.8, 2.4 Hz, 1H) ppm. MS: M/e 135 (M+1)⁺.

Step 2: Synthesis of Intermediate 2

To a stirred solution of benzofuran-5-ol (Intermediate 1, 33.1 kg) and Et₃N (50.8 kg) in DCM (155 kg) was added dropwise a solution of TMSCl(30.4 kg) in DCM (50 kg) at −5 to 0° C. The mixture was warmed to 0˜10° C. and stirred at the temperature for 2 hours (IPC checked INT-1/INT-2=37.4%). The mixture was cooled to between −5 and 0° C. and was added dropwise a solution of TMSCl(10.6 kg) in DCM (8 kg), and then the mixture was warmed to 0˜10° C. and stirred at the temperature for 1 h. The mixture was concentrated below 40° C., and to the mixture was added n-heptane. The mixture was stirred for 20-30 mins and filtered, and the cake was washed with n-heptane. The solvent was distilled out from the filtrate to obtain a crude Intermediate 2 (INT-2%: 62.7%, KF: 0.01%). To a stirred solution of the crude Intermediate 2 above and Et₃N (8.6 kg) in DCM (149 kg) was added dropwise a solution of TMSCl(9.0 kg) in DCM (10 kg) at −5 to 0° C. The mixture was warmed to 0˜10° C. and stirred at the temperature for 1 h (TLC showed the reaction was finished). The reaction mixture was concentrated below 40° C., and to the mixture was added n-heptane. The mixture was stirred for 20-30 mins and then filtered, and the cake was washed with n-heptane. The solvent was distilled out from the filtrate to obtain Intermediate 2 (41.5 kg, INT-2%: 98.1%) as a colorless oil. ¹H NMR (400 MHz, DMSO-d6) δ 7.69 (d, J=2.0 Hz, 1H), 7.21 (d, J=8.8 Hz, 1H), 6.84 (d, J=2.5 Hz, 1H), 6.61 (d, J=2.0 Hz, 1H), 6.56 (dd, J=8.8, 2.5 Hz, 1H), 0.00 (s, 9H) ppm.

Step 3: Synthesis of Intermediate 3

Copper (I) triflate (2:1 complex with toluene, 0.41 kg) and (S, S)-Evans Ligand (0.552 kg) were stirred in DCM (160 kg) at ambient temperature under N₂ atmosphere for 1-2 hours. Intermediate 2 (37.0 kg) was added, followed by a slow addition of ethyl diazoethanoate (58 kg) in DCM (450 kg) at 20˜30° C. The reaction was stirred for 0.5˜1 h at 20˜30° C. (IPC: INT-2/INT-3≤0.2%, residual N₂CHCO₂Et: 0.05%≤1.0%). A solution of EDTA disodium (0.05 mol/L, 150 kg) was added to the reaction mixture for 40˜50min at 20˜30° C. in three times. The organic phase was washed with 25% aqueous NaCl at 20˜30° C. in two times and concentrated below 30° C. The residue was distilled under reduced pressure and crude Intermediate 3 (36.26 kg, 84.5%) was collected at 120˜144° C. The crude compound included the endo- enantiomer which could be removed in the next step. ¹H NMR (400 MHz, DMSO-d6) δ 6.79 (d, J=2.4 Hz, 1H), 6.59 (d, J=8.4 Hz, 1H), 6.42 (dd, J=8.4, 2.4 Hz, 1H), 4.95 (dd, J=5.4, 1.0 Hz, 1H), 3.08 (dd, J=5.4, 3.2 Hz, 1H), 1.02 (dd, J=3.1, 1.2 Hz, 1H), 0.00 (s, 9H) ppm.

Steps 4 and 5: Syntheses of Intermediate 5 and Intermediate 6

To a solution of Intermediate 4 (36.3 kg) in MeOH (108 kg) was added a solution of HCl/MeOH(5M, 0.11 kg) at 20˜30° C., and the mixture was stirred for 2-3 hours (IPC: L/M: 0.5%, chiral purity 90.0%). Et₃N(0.22 kg) was added dropwise at 20˜30° C. The mixture was concentrated and the residue was diluted with n-heptane/EtOAc (4:1) and then concentrated. After adjusting the temperature to 10˜20° C. and stirring for 2˜4 hours at 10˜20° C., the mixture was filtered to give a wet product (Intermediate 5: 94.0%, chiral purity: 90.5%).¹H NMR (400 MHz, DMSO-d6) δ 9.05 (s, 1H), 6.89 (d, J=2.4 Hz, 1H), 6.72 (d, J=8.4 Hz, 1H), 6.55 (dd, J=8.4, 2.4 Hz, 1H), 5.11 (dd, J=5.4, 1.0 Hz, 1H), 3.27 (dd, J=5.4, 3.0 Hz, 1H), 1.19-1.17 (m, 1H) ppm. The crude product was slurried with n-heptane/EtOAc (20:1) three times to give a light yellow solid, which was dried for 12˜16 hours at 40˜50° C. to give 16.55 kg product (Intermediate 6: 98.6%; chiral purity: 99.3%).

Steps 6 and 7: Syntheses of Intermediate 7 and Intermediate 8

To a solution of Intermediate 6 (14 kg) and 5-fluoro-3,4-dihydro-1,8-naphthyridin-2(1H)-one (SM2, 11.2 kg) in DIVIF (66 kg) was added Cs₂CO₃ (26 kg) at 40˜60° C., and the mixture was warmed to 110˜120° C. and stirred for 3 hours at 110˜120° C. The reaction pH was adjusted to 6 with acetic acid (12.0 kg) at 25˜35° C. Water (520 kg) was added and the mixture was stirred for 1˜2 hours. After filtration, the solid was slurried with EA (78 kg) to get a wet product (the purity: (Intermediate 7+Intermediate 8) %: 98%). An aqueous sodium hydroxide solution (125 kg, 2M) was added to a stirred solution of the wet product in THF (240 kg) and stirred for 2˜3 hours at 20˜30° C. (IPC: INT-7/INT-8: 0.9%). The mixture was adjusted to pH 4˜5 with 4N HCl (37 kg) at 20˜30° C. and then stirred for 0.5˜1h. The mixture was concentrated at below 50° C. and a solid precipitated out of the solution. After filtration, the wet product was re-slurried in THF at 35˜45° C. for 1˜2 hours, and then filtered. The resultant wet product was dried for 40 hours at 45˜65° C. to give the title compound Intermediate 8 (18.95 kg: chemical purity 99%, chiral purity 100%). ¹H NMR (400 MHz, DMSO-d6) δ 12.59 (s, 1H), 10.43 (s, 1H), 7.92 (d, J=5.8 Hz, 1H), 7.29 (d, J=2.4 Hz, 1H), 6.97 (d, J=8.8 Hz, 1H), 6.93 (dd, J=8.8, 2.4 Hz, 1H), 6.21 (d, J=5.8 Hz, 1H), 5.21 (dd, J=5.4, 1.0 Hz, 1H), 3.27-3.25 (m, 1H), 2.89 (t, J=7.8 Hz, 2H), 2.51 (d, J=8.8 Hz, 2H), 1.19 (dd, J=3.0, 1.0 Hz, 1H) ppm. MS: M/e 339 (M+1)⁺.

Step 8: Synthesis of Intermediate 9

A solution of Intermediate 8(13.3 kg), DIPEA (16 kg) and HATU (18.1 kg) in DMF (167 kg) was added dropwise into a mixture of 4-(trifluoromethyl)benzene-1,2-diamine (SM3, 7.6 kg) in DMF(74 kg) at 0˜15° C. The mixture was stirred at 20˜25° C. for 4˜6 hours(IPC: INT-9/INT-9: not detected). Active carbon(5.3 kg) in DMF(7.5 kg) was added into the reaction mixture, stirred for 2˜4 hours at 40˜45° C., and then filtered. Water (846 kg) was added dropwise into the filtrate at 15˜30° C., and a solid precipitated out of the solution when stirred for 1˜2 hours. The precipitate was filed and slurried in EtOH at 20˜30° C. for 2-4 hours. After filtration, the wet product was dried for 37 hours at 45˜60° C. to obtain the title compound Intermediate 9 (17.60 kg: 95.5%).

Step 9: Syntheses of free base of Compound 1

A solution of Intermediate 9 (17 kg) and water (1.5 kg) in AcOH(360 kg) was stirred at 65-70° C. for 20 hours (IPC: R/S≤1.0%). The mixture was concentrated to dryness at below 55° C., and active carbon (17 kg) with MeOH(32 kg) was added to the residue. The mixture was stirred for 1 h at about 50° C. After filtration, the filtration was concentrated to remove the solvent at below 45° C. EA(160 kg) and water(330 kg) were added to the residue, followed with an aqueous solution of NaOH(2 mol/L) until pH to 8-9 at 20-30° C. The organic layer was separated, and extracted the aqueous phase with EA. The combined organic phase was washed with water twice, concentrated to dryness to obtain Compound 1 in the form of free base. ¹H-NMR (600 MHz, DMSO-d₆) δ 12.84 (s, 1H), 10.47 (s, 1H), 7.98 (d, J=5.8 Hz, 1H), 7.86 (d, J=1.2 Hz, 1H), 7.69 (m, 1H), 7.48 (t, J=6.2 Hz, 1H), 7.38 (d, J=2.6 Hz, 1H), 7.08 (d, J=8.7 Hz, 1H), 7.02 (dd, J=8.7, 2.6 Hz, 1H), 6.29 (d, J=5.8 Hz, 1H), 5.43 (dd, J=5.4, 1.2 Hz, 1H), 3.55 (dd, J=5.3, 3.3 Hz, 1H), 2.95 (t, J=7.7 Hz, 2H), 2.55 (t, J=7.7 Hz, 2H), 1.97 (d, J=1.3 Hz, 1H) ppm.

Step 10: Syntheses of Compound 1

IPA (83 kg) was added to the residue of Step 9. Maleic acid (5 kg) in water (29 kg) was added into the mixture and stirred for 4 hours at about 50° C., then cooled to 35° C. and stirred for 12 hours at that temperature. The resultant solid was filtered, dried at 40˜60° C., and micronized in a micronizer to give a white powder (Compound 1 Sesqui-Maleate Salt, 8.36 kg) with particle sizes of D90=4.1 μm, D10=1.5 μm, D50=2.4 μm. Compound 1 was identified as a crystalline form by powder X-ray diffraction pattern method as shown in FIG. 4. ¹H-NMR spectra for Compound 1 in the crystalline form is shown in FIG. 5. ¹³C-NMR spectra for Compound 1 in the crystalline form is shown in FIG. 6.

Example 1

Effect of the combination of RAF inhibitor and anti-PD-1-mAb on T cell function in vitro 3D spheroid/PBMC co-culture model

Method

For tumor spheroid generation, HEK-293-OS8-PD-L1 cells and Sk-Mel-28 cells (B-Raf^(V600E)) were mixed at 1:1 ratio at a density of 5×10⁴ cells/mL in ice-cold medium. The Matrigel® Matrix (Corning) was added to the cell suspension at a final concentration of 2% to assist the formation of three-dimensional structures. Cells were plated into ULA 96-well round-bottomed plates (Corning) and grow for three days to form tumor spheroids. Blood samples from healthy donors were collected in heparinized tubes and PBMCs were isolated using Fioll-Paque PLUS reagent (GE Healthcare) according to the instruction of manufacturer. Prior to co-culture with tumor spheroids, PBMCs were activated with 1 μg/mL anti-CD3 antibody for two days. Co-culture was performed in ULA 96-well round-bottomed plates containing one tumor spheroid and 10⁴ PBMCs/well in the present of a series dilution of Compound 1 (5-(((1R,1aS,6bR)-1-(6-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-1a,6b-dihydro-1H-cyclopropa[b]benzofuran-5-yl)oxy)-3,4-dihydro-1,8-naphthyrdin-2(1H)-one Sesqui-Maleate) combining with Mab 1 (containing a heavy chain variable region (Vh) as shown in SEQ ID NO: 24, a light chain variable region (Vk) as shown in SEQ ID NO: 26, and a IgG4 heavy chain effector or constant domain comprising SEQ ID NO: 88) for three days. IFN-γ production level in culture supernatant was determined using an ELISA kit from eBioscience.

Result:

Compound 1 alone promoted IFN-γ releasing from PBMCs in co-culture system at concentration between 10 nM and 100 nM, and suppressed IFN-γ releasing at high concentrations (≥3 μM). Combination of intermediate level Compound 1 and Mab 1 showed significantly enhanced PBMC IFN-γ production, indicating better in vitro T cell activity (FIG. 1).

Example 2

Effect of the combination of anti-PD-1 mAb and RAF inhibitor in a K-Ras mutation lung cancer and B-Raf^(V600E) mutation Colon cancer models in the presence of in the presence of human PBMCs

Method

On the day of implantation, human peripheral blood mononuclear cells (PBMCs) were isolated from 120 mL blood donated by a healthy volunteer. Briefly, peripheral blood was collected into vacuum blood collection tubes containing sodium heparin. PBMCs were separated by density gradient centrifugation using Histopaque-1077 and washed one time by Dulbecco's Phosphate Buffered Saline (DPBS). The cell pellet was suspended with DPBS at appropriate volume to give a final concentration of 1×10⁸ cells/ml and placed on ice prior to inoculation. NOD/SCID mice were pre-treated with cyclophosphamide (prepared in saline, 150 mg/kg, i.p.) and disulfiram (prepared in 0.8% Tween-80 in saline, 125 mg/kg, p.o., one hour after each dose of cyclophosphamide) once daily for two days. Animals were then injected with tumor cells or tumor fragments and PBMCs mixture 24 hours after the second dose of cyclophosphamide.

For Calu-6 K-Ras mutation lung cancer model, the cells were cultured in Dulbecco minimum essential medium (DMEM) supplemented with 10% (v/v) fetal bovine serum, and 100 μg/ml of penicillin and streptomycin. On the day of implantation, culture medium was replaced with fresh medium. Five hours later, media was removed and cells were collected as described above, except that cells were re-suspended in cold (4° C.) DPBS to give a final concentration of 5×10⁷ cells/ml and placed on ice prior to inoculation. Mix the Calu-6 cells, PBMCs and matrigel (BD, Cat#356237) at the ratio of 1:1:2. The right axilla region of each mouse was cleaned with 70% ethanol prior to cell inoculation. Each animal was injected subcutaneously with 5×10⁶ Calu-6 cells and 2.5×10⁶ PBMC (200 μl cell mixture in 50% matrigel) in the right front flank via a 26-gauge needle.

For B-Raf^(V600E) mutation Colon cancer Patient-derived xenograft (PDX) model, BCCO-028 is derived from tumor tissues surgically removed from a patient with colon cancer. Within 2 to 4 hours following patient surgery, the tumor samples were subcutaneously engrafted into the scapular area of anesthetized NOD/SCID mice. After tumors grew to around 300-1000 mm³, tumors were surgically removed and fragments were passaged in NOD/SCID mice by subsequent engraftments. Animals were then implanted with BCCO-028 tumor fragments and PBMCs 24 hours after the second dose of cyclophosphamide. The right axilla region of each NOD/SCID mouse was cleaned with 70% ethanol prior to tumor fragments inoculation. Each animal was implanted subcutaneously with a fragment (around 3×3×3 mm³) of BCCO-028 colon cancer (passage 5) in the right flank via 14-gauge trocar needle, followed by subcutaneously injection of 5×10⁶ PBMC in 200 μl of 50% matrigel near the edge of tumor fragment via a 26-gauge needle.

Starting from day 0 after cell or fragment inoculation, animals were randomly assigned into 4 groups with 8-11 mice per group. The groups consisted of a control group (no drug treatment), 5 mg/kg of Compound 1 (based on free-base weight), 10 mg/kg of Mab 1, and the combination of Compound 1 and Mab 1. Treatments were administered in a volume of 10 ml/kg body weight, assessed immediately before dosing and the volume dosed was adjusted accordingly. Compound 1 was administered by oral gavage (p.o.) once daily (QD) and Mab 1 was administered by intraperitoneal (i.p.) injection once weekly (QW). After implantation, primary tumor volume was measured in two dimensions using a calliper.

Individual body weight was recorded twice weekly, with mice being monitored daily for clinical signs of toxicity for the duration of the study. Mice were euthanized using carbon dioxide once their tumor volume reached 2,500 mm³, the tumor was ulcerated, or body weight loss exceeded 20%.

Tumor volume was calculated using the formula: V=0.5×(a×b²) where a and b are the long and short diameters of the tumor, respectively. Tumor growth inhibition (TGI) was calculated using the following formula:

% TGI=100×[1-treated_(t)/placebo_(t))]

treated_(t)=treated tumor volume at time t

placebo_(t)=placebo tumor volume at time t

Result:

In vivo efficacy of Compound 1 and Mab 1 was examined in BCCO-028 PDX and Calu-6 cells grown subcutaneously in NOD/SCID mice in the presence of human PBMCs. Compound 1 has marked antitumor activity against PDX BCCO-028 colorectal carcinoma (B-Raf^(V600E) mutation) and human Calu-6 lung adenocarcinoma (K-Ras mutation) tumors xenografts in nude mice. In addition, as shown in the figure below, the synergistic efficacy of Compound 1 and Mab 1 is clearly demonstrated by the tumor growth curves in these models. The tumor in the combination-treated group is significantly smaller than either of the monography treatment (FIG. 2, FIG. 3).

The foregoing examples and description of certain embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. All such variations are intended to be included within the scope of the present invention. All references cited are incorporated herein by reference in their entireties.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part of the common general knowledge in the art in any country.

In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprise” or variations such as “comprises” or “comprising” is used in an inclusive sense, i.e., to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The disclosures of all publications, patents, patent applications and published patent applications referred to herein by an identifying citation are hereby incorporated herein by reference in their entirety. 

1. A method for the prevention, delay of progression or treatment of cancer in a subject, comprising administering to the subject in need thereof a therapeutically effective amount of a PD-1 antagonist in combination with a therapeutically effective amount of a RAF inhibitor, wherein the PD-1 antagonist is an antibody or an antigen binding fragment thereof, which specifically binds to human PD-1; and wherein the RAF inhibitor is a compound of Formula (II),

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1, wherein the PD-1 antagonist is an antibody which comprises a heavy chain variable region (Vh) and a light chain variable region (Vk) that contain complement determinant regions (CDRs) listed as follows: a) mu317 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 11, 12, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 14, 15, 16, respectively); b) mu326 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 17, 18, 19, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 20, 21, 22, respectively); c) 317-4B6 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 31, 32, 33, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 34, 35, 36, respectively); d) 326-4A3 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 37, 38, 39, respectively); and CDR-L1, CDR--L2 and CDR-L3 (SEQ ID NOs: 40, 41, 42, respectively); e) 317-1H CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 11, 59, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 14, 15, 16, respectively); f) 317-4B2 CDR-HL CDR-H2 and CDR-H3 (SEQ ID NOs: 11, 60, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 61, 15, 16, respectively); g) 317-4B5 CDR-Hl, CDR-H2 and CDR-H3 (SEQ ID NOs: 11, 60, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 61, 15, 16, respectively); h) 317-4B6 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 11, 32, 13, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 61, 15, 16, respectively); i) 326-1 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 17, 62, 19, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 20, 21, 22, respectively); j) 326-3B1 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 17, 62, 19, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 20, 21, 22, respectively); or k) 326-3G1 CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 17, 62, 19, respectively); and CDR-L1, CDR-I2 and CDR-L3 (SEQ ID NOs: 20, 21, 22, respectively).


3. (canceled)
 4. The method of claim 2, wherein the PD-1 antagonist is an antibody which comprises a heavy chain variable region (Vh) and a light chain variable region (Vk) comprising: a) mu317 (SEQ ID NOs: 4 and 6, respectively); b) mu326 (SEQ ID NOs: 8 and 10, respectively); c) 317-4B6 (SEQ ID NOs: 24 and 26, respectively); d) 326-4A3 (SEQ ID NOs: 28 and 30, respectively); e) 317-4B2 (SEQ ID NOs: 43 and 44, respectively); f) 317-4B5 (SEQ ID NOs: 45 and 46, respectively); g) 317-1 (SEQ ID NOs: 48 and 50, respectively); h) 326-3B1 (SEQ ID NOs: 51 and 52, respectively); i) 326-3GI (SEQ ID NOs: 53 and 54, respectively); j) 326-1 (SEQ ID NOs: 56 and 58, respectively); k) 317-3A1 (SEQ ID NOs: 64 and 26, respectively); l) 317-3C1 (SEQ ID NOs: 65 and 26, respectively); m) 317-3E1 (SEQ ID NOs: 66 and 26, respectively); n) 317-3F1 (SEQ ID NOs: 67 and 26, respectively); o) 317-3G1 (SEQ ID NOs: 68 and 26, respectively); p) 317-3H1 (SEQ ID NOs: 69 and 26, respectively); q) 317-311 (SEQ ID NOs: 70 and 26, respectively); r) 317-4B1 (SEQ ID NOs: 71 and 26, respectively); s) 317-4B3 (SEQ ID NOs: 72 and 26, respectively); t) 317-4B4 (SEQ ID NOs: 73 and 26, respectively); u) 317-4A2 (SEQ ID NOs: 74 and 26, respectively); v) 326-3A1 (SEQ ID NOs: 75 and 30, respectively); w) 326-3C1 (SEQ ID NOs: 76 and 30, respectively); x) 326-3D1 (SEQ ID NOs: 77 and 30, respectively); y) 326-3E1 (SEQ ID NOs: 78 and 30, respectively); z) 326-3F1 (SEQ ID NOs: 79 and 30, respectively); aa) 326-3B N55D (SEQ ID NOs: 80 and 30, respectively); ab) 326-4A1 (SEQ ID NOs: 28 and 81, respectively); or ac) 326-4A2 (SEQ ID NOs: 28 and 82, respectively).


5. The method of claim 2, wherein the PD-1 antagonist is an antibody which contains a IgG4 heavy chain effector or constant domain comprising any of SEQ ID NOs: 83-88.
 6. The method of claim 2, wherein the PD-1 antagonist is an antibody which contains a F(ab) or F(ab)2 comprising a domain comprising the CDRs of claim
 2. 7. The method according to claim 1, wherein the PD-1 antagonist is an antibody which comprises a heavy chain variable region (Vh) and a light chain variable region (Vk), and a IgG4 heavy chain effector or constant domain comprising SEQ ID NOs: 87 or 88, wherein the heavy chain variable region (Vh) and the light chain variable region (Vk) comprise: a) mu317 (SEQ ID NOs: 4 and 6, respectively); b) mu326 (SEQ ID NOs: 8 and 10, respectively); c) 317-4B6 (SEQ ID NOs: 24 and 26, respectively); d) 326-4A3 (SEQ ID NOs: 28 and 30, respectively); e) 317-4B2 (SEQ ID NOs: 43 and 44, respectively); f) 317-4B5 (SEQ ID NOs: 45 and 46, respectively); g) 317-1 (SEQ ID NOs: 48 and 50, respectively); h) 326-3B1 (SEQ ID NOs: 51 and 52, respectively); i) 326-3GI (SEQ ID NOs: 53 and 54, respectively); j) 326-1 (SEQ ID NOs: 56 and 58, respectively); k) 317-3A1 (SEQ ID NOs: 64 and 26, respectively); l) 317-3C1 (SEQ ID NOs: 65 and 26, respectively); m) 317-3E1 (SEQ ID NOs: 66 and 26, respectively); n) 317-3F1 (SEQ ID NOs: 67 and 26, respectively); o) 317-3G1 (SEQ ID NOs: 68 and 26, respectively); p) 317-3H1 (SEQ ID NOs: 69 and 26, respectively); q) 317-311 (SEQ ID NOs: 70 and 26, respectively); r) 317-4B1 (SEQ ID NOs: 71 and 26, respectively); s) 317-4B3 (SEQ ID NOs: 72 and 26, respectively); t) 317-4B4 (SEQ ID NOs: 73 and 26, respectively); u) 317-4A2 (SEQ ID NOs: 74 and 26, respectively); v) 326-3A1 (SEQ ID NOs: 75 and 30, respectively); w) 326-3C1 (SEQ ID NOs: 76 and 30, respectively); x) 326-3D1 (SEQ ID NOs: 77 and 30, respectively); y) 326-3E1 (SEQ ID NOs: 78 and 30, respectively); z) 326-3F1 (SEQ ID NOs: 79 and 30, respectively); aa) 326-3B N55D (SEQ ID NOs: 80 and 30, respectively); ab) 326-4A1 (SEQ ID NOs: 28 and 81, respectively); or ac) 326-4A2 (SEQ ID NOs: 28 and 82, respectively).


8. The method according to claim 1, wherein the PD-1 antagonist is an antibody which comprises a heavy chain variable region (Vh) and a light chain variable region (Vk), and an IgG4 heavy chain effector or constant domain comprising SEQ ID NO: 88, wherein the heavy chain variable region (Vh) and the light chain variable region (Vk) comprises SEQ ID NO: 24 and SEQ ID NO: 26, respectively. 9.-16. (canceled)
 17. The method of claim 1, wherein the RAF inhibitor is a compound of Formula (III),

wherein n is a number from about 0.5 to about 1.5.
 18. The method of claim 1, wherein the RAF inhibitor is a compound of Formula (IIa),


19. The method of claim 1, wherein the cancer is a solid tumor.
 20. The method of claim 1, wherein the cancer or tumor is selected from the group consisting of adrenal cancer, bladder cancer, bone cancer, brain cancer, breast cancer, cervical cancer, colorectal cancer, endometrial cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer (including small-cell lung cancer, or non-small cell lung cancer), lymphoma, melanoma, ovarian cancer, pancreatic cancer, skin cancer, or thyroid tumors and their complications.
 21. The method of claim 20, wherein the cancer or tumor is associated with B-Raf mutations, K-Ras/N-Ras mutations and/or NF1 mutations.
 22. The method of claim 21, wherein the cancer or tumor is non-small cell lung cancer, colorectal cancer, and endometrial cancer, each of which is associated with K-Ras mutations.
 23. The method of claim 1, wherein the PD-1 antagonist and the RAF inhibitor are administered simultaneously, sequentially or separately.
 24. The method of claim 1, wherein the RAF inhibitor is administrated orally at a dose of 5-80 mg QD.
 25. The method of claim 1, wherein the PD-1 antagonist is administered parenterally at a dose of 0.5-10 mg/kg QW, or Q2W, or Q3W, or Q4W.
 26. The method of claim 1, wherein the PD-1 antagonist is an antibody which comprises a heavy chain variable region (Vh) and a light chain variable region (Vk), and a IgG4 heavy chain effector or constant domain comprising SEQ ID NO: 88, wherein the heavy chain variable region (Vh) and the light chain variable region (Vk) comprise SEQ ID NO: 24 and SEQ ID NO: 26, respectively; and the RAF inhibitor is the compound of Formula (IIIa)


27. The method of claim 1, wherein the PD-1 antagonist Mab-1 is administrated at a dose of 0.5-10 mg/kg QW or Q2W or Q3W, and the compound of Formula (Ma) as the RAF inhibitor is administrated at a dose of 5-80 mg QD.
 28. The method of claim 1, wherein the PD-1 antagonist Mab-1 is administrated at a dose of 0.5-10 mg/kg QW or Q2W or Q3W, and the compound of Formula (Ma) as the RAF inhibitor is administrated at a dose of 10-30 mg QD.
 29. A pharmaceutical combination for use in the prevention, delay of progression or treatment of cancer, comprising a PD-1 antagonist and a RAF inhibitor, wherein the PD-1 antagonist is an antibody or a fragment antigen binding thereof, which specifically binds to human PD-1, comprising a heavy chain variable region (Vh) and a light chain variable region (Vk) that contain complement determinant regions (CDRs) listed as follows: CDR-H1, CDR-H2 and CDR-H3 (SEQ ID NOs: 31, 32, 33, respectively); and CDR-L1, CDR-L2 and CDR-L3 (SEQ ID NOs: 34, 35, 36, respectively); and wherein the RAF inhibitor is a compound of Formula (II),

or a pharmaceutically acceptable salt thereof. 30.-56. (canceled) 